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Rational Re-Design of Lactobacillus Reuteri 121 Inulosucrase for Product

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Rational Re-Design of Lactobacillus Reuteri 121 Inulosucrase for Product RSC Advances View Article Online PAPER View Journal | View Issue Rational re-design of Lactobacillus reuteri 121 inulosucrase for product chain length control† Cite this: RSC Adv.,2019,9, 14957 Thanapon Charoenwongpaiboon,a Methus Klaewkla,ab Surasak Chunsrivirot,ab Karan Wangpaiboon,a Rath Pichyangkura,a Robert A. Field c and Manchumas Hengsakul Prousoontorn *a Fructooligosaccharides (FOSs) are well-known prebiotics that are widely used in the food, beverage and pharmaceutical industries. Inulosucrase (E.C. 2.4.1.9) can potentially be used to synthesise FOSs from sucrose. In this study, inulosucrase from Lactobacillus reuteri 121 was engineered by site-directed mutagenesis to change the FOS chain length. Three variants (R483F, R483Y and R483W) were designed, and their binding free energies with 1,1,1-kestopentaose (GF4) were calculated with the Rosetta software. R483F and R483Y were predicted to bind with GF4 better than the wild type, suggesting that these engineered enzymes should be able to effectively extend GF4 by one residue and produce a greater quantity of GF5 than the wild type. MALDI-TOF MS analysis showed that R483F, R483Y and R483W variants Creative Commons Attribution-NonCommercial 3.0 Unported Licence. could synthesise shorter chain FOSs with a degree of polymerization (DP) up to 11, 10, and 10, respectively, while wild type produced longer FOSs and in polymeric form. Although the decrease in catalytic activity and the increase of hydrolysis/transglycosylation activity ratio was observed, the variants could effectively Received 20th March 2019 synthesise FOSs with the yield up to 73% of substrate. Quantitative analysis demonstrated that these Accepted 7th May 2019 variants produced a larger quantity of GF5 than wild type, which was in good agreement with the predicted DOI: 10.1039/c9ra02137j binding free energy results. Our findings demonstrate the success of using aromatic amino acid residues, at rsc.li/rsc-advances position D418, to block the oligosaccharide binding track of inulosucrase in controlling product chain length. This article is licensed under a Introduction oligosaccharide ratio, synthesised by Bacillus megaterium levansucrase could be controlled by site-directed mutagen- Fructooligosaccharides (FOSs) are well-known prebiotics that esis.10,11 Furthermore, rationally designed variants of Bacillus Open Access Article. Published on 14 May 2019. Downloaded 9/27/2021 3:01:54 PM. are widely used in food, beverage and pharmaceutical applica- licheniformis levansucrase can increase the yield of oligosac- tions. FOSs are generally synthesised from sucrose using b- charides (DP4-25) up to 60% of the total products, and also alter fructofuranosidase (E.C. 3.2.1.26) derived from fungi, such as the ratio of isomeric trisaccharide products.12 Aspergillus niger,1,2 Aspergillus japonicas,3,4 Aspergillus aculeatus5,6 Previously, we reported the amino acid residues of Lactoba- and Aspergillus oryzae.7 Bacterial enzymes, such as levansucrase cillus reuteri 121 inulosucrase (LrInu) that play an essential role (E.C. 2.4.1.10) and inulosucrase (E.C. 2.4.1.9), have also been in FOS chain length determination.13 Some variants of this used to synthesise FOSs starting from sucrose producing b-2,6 enzyme, such as D479A, S482A, and R483A, can produce a broad linked levan and b-2,1 linked inulin, respectively.8,9 The bio- range of oligosaccharides with a small amount of accumulating logical activity of FOSs is largely dependent on their degree of polymer, while N543A mainly produces the short chain FOSs polymerization (DP). Many studies have attempted to engineer (DP3-6, with traces of DP $ 7). Since FOSs show optimal these enzymes to increase the yield of FOSs at the expense of prebiotic activity in the DP 2–8 range,14 it is interesting to use macromolecular polymer formation. In the case of levansu- the N543A variant of LrInu as a biocatalyst for the synthesis of crase, for example, the size of levan, as well as the polymer/ such oligosaccharides. However, this variant produced low amounts of transglycosylated product, but liberates high amounts of fructose from sucrose hydrolysis. Therefore, in this aDepartment of Biochemistry, Faculty of Science, Chulalongkorn University, study, we engineered inulosucrase to change its product chain Pathumwan, Bangkok, 10330, Thailand. E-mail: [email protected]; length using computer-aided rational design. The position of [email protected]; [email protected] bStructural and Computational Biology Research Unit, Department of Biochemistry, LrInu selected for mutagenesis was based on the oligosaccha- 13 Faculty of Science, Chulalongkorn University, Pathumwan, Bangkok, 10330, Thailand ride binding track of LrInu reported in previous study. The cDepartment of Biological Chemistry, John Innes Centre, Norwich Research Park, Rosetta program was employed using the homology model of Norwich, NR4 7UH, UK LrInu, built from crystal structure of Lactobacillus johnsonii † Electronic supplementary information (ESI) available. See DOI: inulosucrase (PDB ID: 2YFS),15 to assess variants of LrInu to F, Y 10.1039/c9ra02137j This journal is © The Royal Society of Chemistry 2019 RSC Adv.,2019,9, 14957–14965 | 14957 View Article Online RSC Advances Paper or W, to predict the substrate binding conformations and to noticed that the size of oligosaccharides synthesised by the D compute the binding free energies ( Gbinding) of the substrate/ variants did not correlate well with the distance between the enzyme complexes. The enzyme activities, biochemical prop- mutated sites and catalytic sites. In the case of LrInu, for erties and kinetic parameters of LrInu variants were determined example, the N543A variant mainly produced FOSs with DP3-6, and compared to that of the wild type. Finally, the FOSs while the R483A variant, whose R483 was located close to N543, produced by variant enzymes were analysed by TLC, HPLC and produced the FOSs up to DP12. Although these two residues MALDI-TOF MS. As predicted by Rosetta program, this study were close, the sizes of their FOSs products were dramatically demonstrated the effectiveness of using aromatic amino acids different. This nding suggested that there was more than one to block the oligosaccharide binding track and to control the amino acid residue located in the same binding site that played size of oligosaccharides synthesised by inulosucrase. an important role in substrate binding. Although one of the binding residues was mutated, other residues still could hold Results and discussion the substrate, allowing transglycosylation to occur. Previous study found that blocking the oligosaccharide Rational protein design binding track of levansucrase with aromatic residues (F, Y and Many studies have shown that specicity of carbohydrate- W) was an effective strategy to block elongation of poly- modifying enzymes can be improved by enzyme engineering. saccharide and increase the yields of oligosaccharides.18 The size of glycan produced may be regulated by using two Therefore, we employed this approach in redesigning LrInu so different strategies: removing the interactions between that it could produce high yields of short to medium chain substrates and the enzymes' binding sites or blocking the length oligosaccharides. Because R483 of LrInu is located next substrate binding track with some amino acids, for instance to N543, we hypothesized that blocking at this position might aromatic amino acids (Fig. 1). Previously, many researches increase the yield of short FOSs like that obtained from the demonstrated that a single mutation at some specic amino N543A variant. To test this hypothesis, R483 residue of the Creative Commons Attribution-NonCommercial 3.0 Unported Licence. acid residues of LrInu affected the size of inulin oligosaccharide catalytically competent binding conformation (GF4/Fru-WT) that could be produced.13,16,17 The yield of short-chain oligo- was changed in silico to A, F, Y and W to create GF4/Fru- saccharides was signicantly improved when some amino acid R483A, GF4/Fru-R483F, GF4/Fru-R483Y and GF4/Fru-R483W residues of LrInu were mutated to be alanine, which may reduce complexes, respectively, using the Rosetta program. This cata- the interactions between the enzyme and substrates with lytically competent binding conformation (GF4/Fru-WT) was medium or long chain lengths. This nding has also been re- dened as a binding conformation of 1,1,1-kestopentaose (GF4) ported on other fructosyltransferases, such as Bacillus mega- in the active site of Lactobacillus reuteri 121 inulosucrase con- terium levansucrase11 and Bacillus licheniformis levansucrase.12 taining fru-D272 (also see Experimental section). Since our However, those variants usually produced either very short previous study found that the R483A variant synthesised more This article is licensed under a range of FOSs (DP 3–6) or long range of FOSs (DP $ 12). To GF5 (DP6) than the wild type, the GF4/Fru-R483A complex was control the size of medium range of FOSs at DP # 10 is still used as positive control in this study. Fiy independent runs of challenging for the eld of enzyme engineering and food the FastDesign protocol were employed to resolve unfavorable Open Access Article. Published on 14 May 2019. Downloaded 9/27/2021 3:01:54 PM. industry. Therefore, in this study, inulosucrase was redesigned interactions and nd low energy binding conformations of all D by computationally assisted method in order to increase the complexes (Fig. 2). Gbinding of each binding conformation was specicity
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